The present invention relates generally to the field of transdermal extraction and sampling of analytes. More particularly, the present invention provides a method for extraction and sampling of analytes by electroporation in the presence of liposomes.
The ability to accurately measure analytes such as glucose in the blood is important for management of disease. For example, proper monitoring of blood glucose levels prior to or following insulin injections could result in easier management of diabetes.
Conventional monitoring of blood analytes requires drawing of a blood sample. However, pain and inconvenience associated with frequent drawing of blood may cause patients to miss monitoring routines. To overcome problems associated with invasive techniques, several approaches have been proposed that involve extraction of analytes through skin.
Mammalian skin has two layers, the epidermis and the dermis. The epidermis is a stratified squamous keratinizing epithelium. The uppermost stratum of the epidermis is the stratum corneum (SC) which consists of about 20 layers of flattened, enucleate, keratin-filled corneocytes surrounded by lamellae of about 8 lipid bilayers on average. The bilayers consist primarily of cholesterol, free fatty acids and ceramide. The total thickness of the SC varies from 10 to 40 xcexcm, with an average thickness of 20 xcexcm (Chizmadzhev et al., 1995, Biophysical Journal, 68:749-765; Bouwstra et al., 1995; Pharmaceutical Research 11 (12):1809-1814; Swartzendruber et al., 1989, Journal of Investigative Dermatology, 92:251-257). The SC layer constitutes the major electric resistance of the skin (Rosendahl, 1945, Acta Physiol. Scan. 9:39), and is the main barrier to substance transport. Thus, transdermal extraction approaches have typically involved techniques which increase permeability of the SC layer. Examples of such techniques are iontophoresis, ultrasound, and vacuum.
Iontophoresis requires the use of electrodes containing oxidation-reduction species as well as passing electric current through the skin. A DC field is used to transport molecules through the SC via appendageal or paracellular space. However, the non-permeable nature of SC and the side effects associated with iontophoresis such as irreversible skin damage, limit the use of iontophoresis for transdermal sampling.
Vacuum has been used to draw fluid transdermally for sampling (U.S. Pat. No. 6,009,343). However,.the impermeability of the stratum corneum layer limits the use of this technique.
Ultrasound is believed to disrupt the lipid layers between the keratinocytes in the stratum corneum and thereby improve permeability of the skin. Methods involving ultrasound are disclosed in U.S. Pat. Nos. 4,767,402; 4,780,212 and 5,115,805.
Another method to transiently permeabilize skin is by the application of a single or multiple short duration pulses. The predominant voltage gradient across the skin develops across the non-conductive SC. If the voltage gradient exceeds the barrier breakdown potential, pores are formed which reseal depending on the applied pulse field and duration (Hui, 1995, In: Methods in Molecular Biology, Vol:48 Animal cell Electroporation and Electrofusion Protocols, J. A. Nickoloff, ed. Humana Press, pp. 29-40). During the lifetime of the pores, materials may be transported across the barrier (Powell et al., 1989, Biophysical Journal 56:1163-1171; Prausnitz et al.,1993, Proc. Nat. Acad. Sci., USA 90:10504-10508). While electroporation has been used for delivery of materials, it has not been used heretofore for the extraction and sampling of body analytes such as glucose.
Although liposomes have been used for topical transdermal drug administration with various degrees of effectiveness, the mechanism is still debatable (Mezei, 1988, In: Liposomes as drug carriers, Gregoriadis G. Ed.,Wiley, New York). When applied to the histocultured murine skin surface, neutral liposomes were reported to concentrate in the hair follicle channels (Li et al., 1992, In Vitro Cell Dev. Biol. 28A:679-681; Li et al., 1993, In Vitro Cell Dev. Biol. 29A:258-260). Based on the above, the role of liposomes in analyte extraction and sampling is not clear. Moreover, no quick and efficient techniques exist for extraction and analysis of blood analytes. Consequently, there is an ongoing need to develop new approaches for extraction and sampling of analytes so as to allow easy monitoring and management of diseased states.
The present invention provides a method for transdermal sampling of analytes. The method comprises forming a local area of permeabilized skin by contacting the skin with liposomes comprising negatively charged lipids, and passing an electric pulse through the permeabilized skin. Extracellular fluid is then collected by application of suction in the permeabilized area. The collected fluid can then be used for detecting the presence of desired analytes.
The negatively charged lipids may be mixed with other negatively charged or uncharged lipids to form liposomal compositions useful for the present invention. Thus, in a preferred embodiment, liposomes were formed by negatively charged phospholipid dioleylphosphatidylglycerol (DOPG) with and without uncharged dioleylphosphatidyl choline (DOPC).